There is a significant need for development of alternative strategies for targeted kinase inhibition. As an alternative for selective kinase targeting, this study is focused on the development of chemically constrained peptides that target allosteric activation mechanisms. This proposal is the crucial first step in the development of a powerful new tool towards developing peptide-based allosteric inhibitors of kinases. The long-term goal of this work is to develop allosteric effectors that target kinase activation as it relates to cancer. The overall objective for this application is to develop novel, peptide-based compounds that disrupt protein-protein interactions (PPIs) involved in allosteric activation of EGFR. It is the central hypothesis that by targeting alternative conserved surfaces of EGFR that regulate PPIs rather than the ATP or EGF binding sites, EGFR can be effectively inhibited and this targeting strategy may retain activity in patients that are unresponsive to current EGFR-targeted therapies. The rationale underlying the proposed project is that the knowledge gained by developing allosteric EGFR inhibitors has the potential to translate into innovative, highly specific targeting strategies for successful inhibition of diverse kinases in cancer. This hypothesis will be tested by pursuing two specific aims: 1) Evaluate inhibitory properties of constrained EGFR dimerization arm peptides in different classes of resistance to EGFR-targeted therapies; and 2) Optimize inhibitory properties of constrained peptides mimicking the dimerization arm of EGFR. The expected outcomes of this work will be significant. First, this study will validate the concept that allosteric kinase activation mechanisms can be targeted for inhibition. Next, it will be determined whether these alternative targeting approaches can inhibit lung cancer cell lines that either differ in EGFR expression levels or are resistant to EGFR-targeted therapies by monitoring formation of ErbB homo- and heterodimers. ErbB inhibition will be extensively characterized in cell lines with various resistance mechanisms. In silico approaches will also be applied to optimize ligand binding and efficacy and will validate the compounds biochemically. Ultimately, the synthetic strategies developed in this study can also be applied to diverse protein-protein interaction surfaces. This work will contribute significantly to our understanding of designing compounds that target kinase allostery. The research proposed in this application is innovative because it represents a new and substantive departure from the status quo by proposing that PPIs play a significant role in kinase activation and therefore allosteric activation mechanisms can be targeted using alternative synthetic strategies including novel compounds included in this study that target and inhibit the PPI interface. This contribution is significant because it will undoubtedly reveal new, alternative approaches for inhibition of EGFR and other receptor tyrosine kinases.